1. Field of the Invention
The present invention relates to a test which can be used to predict pre-eclampsia in pregnant women.
2. Related Art
Pre-eclampsia is a disorder of human pregnancy. Which affects around 5 to 10% of pregnancies. The underlying cause of pre-eclampsia remains unclear in spite of extensive clinical and basic research. Pre-eclampsia is the definition given to the condition in pregnancy in which elevated blood pressure is associated with proteinuria. Pre-eclampsia is distinct from eclampsia which is additionally associated with convulsions. Pre-eclampsia is defined in Souhami and Moxham Textbook of Medicine, Second edition, Churchill Livingstone (1994), as an abnormal rise in blood pressure between the first and second halves of pregnancy of xe2x89xa630/20 mmHg, with abnormal urate levels of  greater than 0.35 mmol/l at 32 weeks or  greater than 0.4mmol/l thereafter, associated with proteinuria, impaired renal function and clotting disorders. The consequences of pre-eclampsia are serious and include reduced uteroplacental perfusion, foetal growth retardation, pre-term birth, and increased foetal and maternal morbidity and mortality.
There have been many attempts to provide a reliable predictive test for pre-eclampsia. Previous suggestions have involved assays for the levels of circulating biochemical markers in the mother""s blood but to date the scientific literature on this issue is contradictory and inconclusive. The following hormones have all been identified as possible markers in an elevation of levels might be predictive of pre-eclampsia in maternal plasma: progesterone, oestradiol, total human chorionic gonadotrophin (hCG), corticotrophin-releasing factor (CRF), adrenocorticotrophin (Muller et al Am. J. Obst. Gynecol. 175 37-40 (1996); Ashour et al Am. J. Obst. Gynecol. 176 438-444 (1997); Hsu et al Am. J. Obst. Gynecol. 170 1135-1138 (1994); Wenstrom et al A. J. Obst. Gynecol. 171 1038-1041 (1994)). Conversely, levels of oestriol, human placental lactogen and cortisol are unchanged or decreased. Whilst circulating CRF has been proposed as a prognostic marker for pre-eclampsia, treatment of hypertension does not influence maternal CRF levels and nor has any correlation been found between CRF levels and mean blood pressure.
Other possible markers which have been suggested are Activin A and Inhibin A. Activin is a hypophysiotrophic factor produced by the placenta which is know to act as a growth factor having activity in modulating cell growth and differentiation. Currently, there are three forms of activin which are recognised to exist as homodimeric proteins: Activin A (xcex2Axcex2A). Activin AB (xcex2Axcex2B) and Activin B (xcex2Bxcex2B) in which the subunits are linked by disulphide bridges. Inhibins are heterodimeric proteins consisting of xcex1xcex2A (Inhibin A) and xcex1xcex2B (Inhibin B) subunits also linked by disulphide bridges. Additionally monomeric Inhibin xcex1 subunits are present in the circulation and follicular fluid. Inhibin is thought to have an endocrine role which inhibits pituitary production of follicle-stimulating hormone (FSH). Muttikrishna et al (The Lancet 349 1285-1288 (1997)) have proposed that Activin A and Inhibin A might be suitable markers for the onset of pre-eclampsia. These proteins were suggested because they were thought to be more sensitive markers than hCG or corticotrophin-releasing hormone where there is a considerable overlap in elevated hormone levels between control and pre-eclamptic women.
However, it has now been found that a predictive test for pre-eclampsia which is based on levels of free xcex2-human chorionic gonadotrophin (free xcex2-hCG) and Inhibin A can in fact provide a surprisingly improved level of predictiveness over previously known tests. The present invention describes a system of screening for pre-eclampsia, in which a single risk estimate is derived from measurements carried out on biochemical samples obtained during pregnancy.
According to a first aspect of the invention there is provided a method of predicting the risk of pre-eclampsia in a pregnant woman, the method comprising the steps of:
(a) obtaining a sample of blood from the woman;
(b) subsequently assaying the sample for the levels of free xcex2-human chorionic gonadotrophin (free xcex2-hCG) and Inhibin A present in the sample; and
(c) determining the risk of pre-eclampsia using the measured levels of free xcex2-human chorionic gonadotrophin (free xcex2-hCG) and Inhibin A present in the sample.
Methods according to the present invention are carried out ex vivo. In the step (a), the sample of blood may be collected by any suitable means from the pregnant woman. The species free xcex2-human chorionic gonadotrophin (free xcex2-hCG) is distinct from the intact or total form of the molecule which is referred to simply as hCG or total hCG. The assay of the sample in step (b) for the levels of free xcex2-human chorionic gonadotrophin (free xcex2-hCG) and Inhibin A present in the sample may be carried out using standard protocols e.g. those based on ELISA (Enzyme-Linked ImmnoSorbent Assay) or RIA (RadioImmunoAssay), or commercially available kits, e.g. free xcex2-human chorionic gonadotrophin (free xcex2-hCG) can be measured using the solid phase, two site fluoroimmunometric assay based on a direct sandwich technique as described by Stevenson et al (Ann. Clin. Biochem. 30 99-100 (1993)) and Spencer et al (Ann. Clin. Biochem. 29 506-518 (1992)). Inhibin A can be measured according to the solid phase sandwich ELISA assay described by Groome et al (J. Immunol. Methods 165 167-176 (1993); Clin. Endocrinol., 40 717-723 (i994)). In particular embodiments of the present invention, the assay may also comprise an analysis of the levels of unconjugated oestriol (uE3), which can be measured using the solid phase, time resolved fluoroimmunoassay described in U.S. Pat. No. 4,565,790 and U.S. Pat. No. 4,808,541. Additionally, since free xcex2-hCG and total hCG are known to be highly correlated in pregnancy, total hCG may also be used as a screening marker for pre-eclampsia in a method according to the present invention as an alternative to free xcex2-hCG. The intact hCG molecule (total hCG) can be measured directly using exactly the same method as for the free xcex2-subunit with AFP, i.e. sold phase, two-site fluoroimmunometric assay based on a direct sandwich technique. Preferably, the markers used are free xcex2-hCG and Inhibin A measured after 20 weeks of pregnancy, and particularly at the end of the second trimester and the beginning of the third trimester.
In step (c), the determination of the risk of pre-eclampsia can be undertaken by comparing the levels of free xcex2-human chorionic gonadotrophin (free xcex2-hCG) and Inhibin A present in the sample with those in a control sample, or the median level in a group of control samples, i.e. samples from unaffected pregnancies, to provide a prediction of the probability of the onset of pre-eclampsia in the woman. The determination of risk nay comprise deriving the likelihood ratio using a multivariate analysis based on distribution parameters from a set of reference data.
Calculation of risk from the measured marker levels is based on the observed relative frequency distribution of marker levels in (a) pre-eclamptic and (b) unaffected pregnancies. Any of the known statistical techniques may be used. Preferably the multivariate Gaussian model is used, which is appropriate where the observed distributions are reasonably Gaussian. Such multivariate Gaussian analysis is in itself known, for example from Wald N J, Cuckle H S, Densem J W, et al (1988); Maternal serum screening for Down""s syndrome in early pregnancy. BMJ 297, 883-887 and Royston P, Thompson S G (1992); Model-based screening by risk with application to Down""s syndrome. Stat Med 11, 256-268.
In a preferred method, two Gaussian heights are calculated, (a) one for the pre-eclamptic distribution and (b) the other for the unaffected distribution. For this calculation, the necessary statistical parameters which specify the Gaussian distributions are the mean, the standard deviation and the correlations for the two distributions. The distributions are stored as reference data for use in analysis. The ratio of the two Gaussian heights gives the likelihood ratio which is a measure of the increased risk of having a disorder, given a particular combination of lest results, compared to the background risk, i.e. the risk in the absence of test results.
The estimation of risk consists of multiplying the likelihood ratio by the background risk for pre-eclampsia. The estimated risk is classified as screen-positive or screen negative based on a comparison with a predetermined risk cut-off. The value of the risk cut-off may be altered to vary the detection rate and false positive rate.
Methods in accordance with the present invention may further comprise the step (d) of re-expressing each measured screening marker level as a multiple of the median level of the respective screening marker in unaffected pregnancies of the same gestational age as the fetus of the pregnant woman. The screening marker levels may also be adjusted to allow for one or more factors selected from the group of maternal race, maternal weight, multiple birth and diabetic status.
According to a second aspect of the present invention there is provided an apparatus for determining whether a pregnant woman is at an increased risk of pre-eclampsia, the apparatus comprising:
(a) data input means for inputting a measurement of the serum levels of Inhibin A and free xcex2-human chorionic gonadotrophin (free xcex2-hCG) in a sample obtained from said pregnant woman; and
(b) calculation means for determining the risk of pre-eclampsia using the input levels of the serum markers Inhibin A and free xcex2-human chorionic gonadotrophin (free xcex2-hCG).
In certain embodiments of the invention, the calculation means may be arranged to determine the risk of pre-eclampsia by deriving the likelihood ratio for pre-eclampsia using a multivariate analysis based on distribution parameters derived from a set of reference data. Preferably the multivariate analysis is a multivariate Gaussian analysis.
Apparatus in accordance with this aspect of the invention may further comprise (c) means for re-expressing the levels of each input screening marker Inhibin A and free xcex2-human chorionic gonadotrophin (free xcex2-hCG) as a multiple of the median level of the respective screening marker in unaffected pregnancies of the same gestational age as the fetus of the pregnant women and supplying the re-expressed screening marker levels to said calculation means.
According to a third aspect of the present invention there is provided a method of operating an apparatus as described in accordance with the second aspect to determine the risk of pre-eclampsia in a pregnant woman. The data input means may be used to enter items of data identified as the concentrations of serum markers Inhibin A and free xcex2-human chorionic gonadotrophin (free xcex2-hCG) in a sample obtained from a pregnant woman. The calculation means may be used to calculate the risk of pre-eclampsia using the input levels of the serum markers. The operation the different steps and preferred features are as described above. In another preferred embodiment of this aspect of the invention, the method comprises the steps described in FIGS. 4, 5, 6 and 7.
According to a fourth aspect of the invention there is provided a kit for predicting the onset of pre-eclampsia in a pregnant woman, comprising means for assaying a sample from the women for the levels of free xcex2-human chorionic gonadotrophin (free xcex2-hCG) and Inhibin A present in the sample.
Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis. 